APPLICANT'S ABSTRACT: In the previous stage of this project, supported by DA 03025-07 through 09 we have studied the effects of cocaine in tolerance model involving continuous administration of cocaine to rats through osmotic minipumps, the possible involvement of voltage-dependent sodium channels in the development of seizure activity upon repeated local anesthetic treatment, and the activity of cocaine and its congeners in model systems for the plasma membrane and vesicular dopamine (DA) transporter. In the cocaine tolerance model, we focused on the interaction between DA and serotonin (5-HT) circuits with measurements of ln vitro release of DA and 5-HT in DAergic cell body and terminal areas. In the present application, we propose to test hypotheses developed on the basis of the previous experiments with in vivo measurements of extracellular DA and 5-HT by microdialysis. Because chronic cocaine treatment changes the effect of acute cocaine challenge, it is important to further characterize the acute effect. Cocaine will be applied focally through a dialysis probe in the VTA, and extracellular DA, 5-HT, and norepinephrine (NE) will be determined simultaneously in dialysates from this probe and a second probe in the NACC. The involvement of DA autoreceptor regulation and 5-HT uptake blockade will be assessed by the use of autoreceptor agents and lesioning techniques. The potential involvement of K+ channel in autoreceptor regulation in the VTA will be studied with quinine by both in vivo and in vitro approaches.The effect of focal application of selective blockers of 5-HT, DA, and NE uptake and a sodium channel blocker will be compared with that of cocaine (an aselective amine uptake and sodium channel blocker) on extracellular amines in the VTA and NACC. Animals will be made tolerant to the locomotor activating effect of cocaine by continuous administration via osmotic minipumps or sensitized by intermittent injections, and extracellular DA, 5-HT, and NE will be measured simultaneously in dialysates from probes in the VTA and NACC. The effect of challenge with cocaine will be measured, as well as the response to autoreceptor agents. A potential regulation of K+ channels linked with autoreceptors will be studied with quinine. In addition, experiments will address potential changes in GABAergic or glutamatergic systems known to impinge on mesolimbic DA neurons. If GLU-related changes are found, the potential implication of nitric oxide (N0) as a messenger in the effect on DA release will be studied by the use of N0 synthase activators and inhibitors, initially in in vitro experiments. In addition, we wish to extend our studies on the effect of cocaine in model systems for DA storage. Preparations of rat brain synaptic vesicles and bovine chromaffin granules will be used to study the structure-activity relationships of cocaine congeners in inhibiting DA transport and [3H]dihydrotetrabenazine binding, and a comparison will be made with those for interacting with the DA transporter in the plasma membrane. Experiments will address factors other than binding of cocaine to the vesicular transporter, such as the potential effects on Ph (alkalinization of vesicle interior) or membrane potential (depolarization). If cocaine turns out to be a pure blocker of uptake by binding to the transporter, we will address the question of whether the site that binds cocaine (or other uptake blockers such as dihydrotetrabenazine, ketanserin, and amphetamine) and the site that binds amine substrates (DA, NE, 5-HT) are identical, overlap, or otherwise interact (allosterically?) through equilibrium and kinetic binding studies and irreversible blockade.